Many of the proteins found in pathological protein fibrils also exhibit tendencies for liquid-liquid phase separation (LLPS) both in vitro and in cells. The mechanisms underlying the connection between these phase transitions have been challenging to study due to the heterogeneous and dynamic nature of the states formed during the maturation of LLPS protein droplets into gels and solid aggregates. Here, we interrogate the liquid-to-solid transition of the low complexity domain of the RNA binding protein FUS (FUS LC), which has been shown to adopt LLPS, gel-like, and amyloid states. We employ magic-angle spinning (MAS) NMR spectroscopy which has allowed us to follow these transitions in real time and with residue specific resolution. We observe the development of ?-sheet structure through the maturation process and show that the final state of FUS LC fibrils produced through LLPS is distinct from that grown from fibrillar seeds. We also apply our methodology to FUS LC G156E, a clinically relevant FUS mutant that exhibits accelerated fibrillization rates. We observe significant changes in dynamics during the transformation of the FUS LC G156E construct and begin to unravel the sequence specific contributions to this phenomenon with computational studies of the phase separated state of FUS LC and FUS LC G156E.

中文翻译：

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FUS LC液固转变过程中结构和动力学的实时观察

在病理性蛋白原纤维中发现的许多蛋白在体外和细胞中也表现出液-液相分离（LLPS）的趋势。由于LLPS蛋白小滴成熟为凝胶和固体聚集体时形成的状态具有异质性和动态性，因此研究这些相变之间的联系所依据的机理一直具有挑战性。在这里，我们询问了RNA结合蛋白FUS（FUS LC）的低复杂性结构域的液-固过渡，该结构已显示采用LLPS，凝胶样和淀粉样蛋白状态。我们采用了魔角旋转（MAS）NMR光谱技术，该技术使我们能够实时且以残留物特定的分辨率跟踪这些转变。我们观察到在成熟过程中α-片层结构的发展，并表明通过LLPS产生的FUS LC原纤维的最终状态不同于原纤维种子生长的状态。我们还将我们的方法应用于FUS LC G156E，这是一种临床相关的FUS突变体，具有加速的原纤维形成速率。我们观察到在FUS LC G156E构建体转化过程中动力学发生了重大变化，并通过对FUS LC和FUS LC G156E的相分离状态进行了计算研究，开始揭示了对该现象的序列特异性贡献。